Macromolecular translocation across prokaryotic and eukaryotic membranes is a major area of biomedical interest. In recent years, secretion systems ancestrally-related to bacterial flagellar and conjugation systems have been shown to play important roles in pathogenesis by translocating effector molecules to the eukaryotic cell cytosol during the course of infection. These systems are now designated as types III and IV secretion pathways, respectively. The focus of work in this laboratory is the type IV transfer system used by A. tumefaciens to deliver oncogenic T-DNA to susceptible plant cells. The T-DNA transfer system is an excellent model for detailed mechanistic studies of type IV secretion. Recent work has shown that the T-DNA transfer system is exceptionally versatile both in terms of substrate selection and target cell recognition. In addition to the T-DNA secretion substrate, this system can export other DNA substrates as well as effector proteins independently of DNA. Furthermore, this system can translocate substrates to other bacteria and to a wide variety of eukaryotic cell types, including those of plants, fungi, and humans. The overall goals of work in this laboratory are to: i) define the assembly pathway for this type IV transfer system, ii) characterize its architectural arrangement, iii) elucidate the reaction mechanisms underlying substrate processing and delivery to the secretion channel, and iv) define the route of translocation across the Gram-negative bacterial envelope. It is now established that substrate transfer requires a pilus for mediating cell-cell contacts and a channel for translocation across the bacterial cell envelope. We will use a combination of molecular, genetic, and biochemical approaches in the following specific aims. First, we will explore structure - function relationships of the inner membrane VirB11 ATPase to define its role in transporter biogenesis and substrate translocation; for these studies we will capitalize on our assemblage of a large collection of altered-function mutants and a crystal structure of a VirB11 homolog. Second, we will identify steps in the substrate delivery pathway, focusing on the model VirE2 substrate and the VirE1 secretion chaperone. Finally, we will characterize critical steps in the assembly pathway of this transfer system; these studies will identify specific contacts involving three proteins essential for transporter assembly and structural integrity, the polytopic inner membrane protein VirB6 and two outer membrane proteins, VirB7 lipoprotein and VirB9.